Identification of faecal Escherichia coli isolates with similar patterns of virulence and antimicrobial resistance genes in dogs and their owners

Abstract Background The presence of antimicrobial resistance and virulence genes in Escherichia coli allows them to survive and cause infections. The close contact between humans and pets can reinforce the risk of transmitting resistant and virulent bacteria between them. Objectives This study aims to compare the patterns of the presence of tetracycline and streptomycin resistance genes, as well as important virulence genes in E. coli isolated from faeces of healthy dogs and their owners. Methods Polymerase chain reactions were performed for detection of antimicrobial resistance (tetA, tetB, tetC, tetD, strA and strB) and virulence (fimH, iss, sitA and malX) genes in 144 faecal E. coli isolates from 28 dog–owner pairs and 16 humans who did not keep any pets as controls. Results Among the investigated antimicrobial resistance and virulence genes, tetA (52.1%) and fimH (86.8%) genes had the highest prevalence. No statistically significant difference was found between the prevalence of antimicrobial resistance and virulence genes in isolates of dogs and their owners. In total, 46.4% of dog–owner pairs had the same patterns of presence or absence of six antimicrobial resistance genes, 50.0% had the same patterns of presence or absence of four virulence genes and 25.0% had the same patterns of presence or absence of all 10 tested genes. Conclusion The presence of antimicrobial‐resistant virulent E. coli in humans and pets may predispose them to infections that are hard to cure with conventional antibiotics. Notable frequency of dogs’ and their owners’ E. coli isolates with similar patterns of antimicrobial resistance and virulence genes may indicate the possibility of sharing virulent antimicrobial resistant E. coli between them.


INTRODUCTION
Escherichia coli are well-known commensal bacteria of the gastrointestinal tract of humans and animals, including dogs (I. Carvalho et al., 2021). As an opportunistic pathogen, E. coli can cause intestinal and extra-intestinal diseases (Abreu-Salinas et al., 2020;I. Carvalho et al., 2021). It can be shed in faeces and spread easily through feed, water and soil. Moreover, the zoonotic transmission of E. coli between humans and companion animals is possible (Massella et al., 2021;Qekwana et al., 2018).
Escherichia coli can acquire antimicrobial resistance genes from the same or different bacterial species, through horizontal gene transfer.
Also, the extensive use of antibiotics in humans and animals can cause antibiotic resistance and the selection pressure on this normal flora (Skurnik et al., 2016;Yasugi et al., 2021).
In addition to the antimicrobial resistance genes, the presence of different virulence factors in E. coli allows them to survive and cause infections. There are several known virulence genes in E. coli including, adhesins such as type-1 fimbriae (fimH), protectins such as increased serum survival (iss), sidrophores such as the iron transporter sitA and the pathogenicity islands marker (malX) with glucose and maltose transporting activity (Naziri et al., 2020).
Like humans, dogs' intestines also can be reservoirs of commensal and pathogenic E. coli strains, which carry different antibiotic resistance and virulence genes. So, a human can acquire these strains through direct contact with dogs and their faeces, and also indirectly from common environments (Naziri et al., 2015). As the number of humans who keep dogs is increasing worldwide, this close contact can reinforce the risk of sharing resistant and virulent bacteria between dogs and their owners (Abreu-Salinas et al., 2020;I. Carvalho et al., 2021). Therefore, the objective of the current study was to determine and compare the patterns of the presence of four tetracycline and two streptomycin resistance genes and also four important virulence genes in E. coli strains isolated from faeces of healthy dogs and their owners.

Bacterial strains
A total of 144 confirmed E. coli strains investigated in the current study were previously isolated from faecal samples of 28 healthy dogs (two isolates from each; n = 56), 28 healthy dog owners (two isolates from each; n = 56) and 16 healthy humans who did not keep any pets as controls (two isolates from each, n = 32; Naziri et al., 2015). All human volunteers who participated in the sampling were over 18 years old; they filled out and signed the informed consent.

Antimicrobial resistance and virulence factors genes detection
In order to detect the presence of four tetracycline resistance genes (tetA, tetB, tetC and tetD) and two streptomycin resistance genes (strA and strB) and also to investigate the presence of four virulence genes including fimH, iss, sitA and malX, polymerase chain reactions (PCRs) were performed on DNA of 144 E. coli isolates that were extracted by the boiling method (Derakhshandeh et al., 2014). The primer sequences, amplicon size and PCR conditions were summarised in Table 1. Finally, the PCR products were electrophoresed on 1% agarose gel (Parstous) containing a safe stain (YTA) and visualised using a UV-transilluminator (UVitec).

Statistical analysis
Comparisons of the prevalence of antimicrobial resistance and virulence genes in E. coli isolates of the different studied groups were performed by the Pearson chi-square (χ2) test and Fisher's exact test (SPSS 16.0,SPSS Inc.). A value of P ≤ 0.05 was regarded as statistically significant.
coli isolates. The prevalence of tetracycline resistance genes (tetA, tetB, tetC and tetD) and streptomycin resistance genes (strA and strB) among E. coli isolates of three studied groups (dogs, dog owners and control humans) is shown in Table 2.
The prevalence of four tetracycline and two streptomycin resistance genes was not significantly different in isolates of dogs, dog owners and control humans (P > 0.05). Moreover, no significant difference (P > 0.05) was found in the prevalence of these resistance genes between females and males isolates in each groups, except for the tetB gene, which was more prevalent (P = 0.028) in female control humans isolates, and the strA gene, which was more prevalent in male owners isolates (P = 0.055).
Comparison of patterns of the antimicrobial resistance genes in E.  Statistical analysis revealed that the prevalence of four studied virulence genes was not significantly different in isolates of dogs and their owners (P > 0.05). The iss and sitA genes were significantly more prevalent in the E. coli isolates of dogs and dog owners than in control humans' (P < 0.05). Only the prevalence of malX gene was significantly higher in control humans' isolates than dogs' (P = 0.003) and dog owners' (P = 0.007) isolates ( Figure 2).
Moreover, no significant difference (P > 0.05) was found in the prevalence of these four virulence genes between females' and males' isolates in each groups, except for the fimH gene, which was more prevalent (P = 0.051) in male dogs' isolates, and the iss gene, which was more prevalent in female owners' isolates (P = 0.042).  In I. Carvalho et al.'s (2021) study, a significant prevalence of tetA or tetB genes was found among most of the tetracycline resistant E. coli

DISCUSSION
isolates. In their study, the prevalence of tetracycline-resistant E. coli was 80% and 90% in healthy and sick dogs, respectively. Furthermore, the prevalence of streptomycin-resistant E. coli was 60% and 40% in healthy and sick dogs, respectively (I. Carvalho et al., 2021). Karahutová et al. (2021) stated that healthy dogs can be a potential reservoir of resistant bacteria. In their study, antimicrobial resistance genes were more frequent in E. coli isolates of healthy dogs than in diarrhoeic dogs. The highest frequency of antimicrobial resistance in faecal E. coli isolates of both healthy and diarrhoeic dogs was against tetracycline (34.21% and 31.11%, respectively). The tetA gene (50%) was more prevalent in healthy dogs, while the tetB gene was only detected in 13.18% of the E. coli isolates from healthy dogs. On the contrary, in diarrhoeic dogs, the prevalence of tetB gene (28.89%) was higher than tetA gene (11.11%; Karahutová et al., 2021).
In a similar study conducted by A. C. Carvalho et al. (2016) on the E. coli isolates from dogs and their owners, the prevalence of streptomycin and tetracycline resistance in dogs' isolates was 66.6% and 50.0%, respectively. Moreover, the prevalence of streptomycin and tetracycline resistance in owners' isolates was 64.3% and 52.3%, respectively. In their study, the highest frequency of resistance was recorded against ampicillin, tetracycline, streptomycin and trimethoprim-sulfamethoxazole (A. C. Carvalho et al., 2016).
Besides the antimicrobial resistance genes that have an important role in the survival of bacteria, the virulence genes also have a key role in the pathogenesis of bacteria (Massella et al., 2021).
Escherichia coli have more than 50 genes that encode adhesins, toxins, siderophores, invasins, protectin, capsular antigens and miscellaneous virulence genes that give them the ability of attaching and colonising the host cells, acquisition of iron, toxicity, pathogenicity and so forth (Flament-Simon et al., 2020;Naziri et al., 2020).
Variable frequencies of virulence factors and antimicrobial resistance genes can be due to differences in strains of studied E. coli in various geographical regions.

CONCLUSION
The presence of antimicrobial-resistant virulent E. coli in humans and pets may predispose them to infectious diseases that are hard to cure with conventional antibiotics. Moreover, the notable frequency of E. coli isolates with similar antimicrobial resistance and virulence genes patterns in dogs and their owners may indicate the possibility of sharing the virulent antimicrobial-resistant E. coli between them.

AUTHOR CONTRIBUTIONS
Study design and acquisition of funding: Abdollah Derakhshandeh.

ACKNOWLEDGEMENTS
We acknowledge all the volunteer participants, the small animal veterinary clinics of Shiraz, Iran, particularly Shiraz Mehr clinic and Dr. Aidin Shojaee Tabrizi, for their cooperation in sampling.

DATA AVAILABILITY STATEMENT
Further data are available from the corresponding author upon reasonable request.

ETHICS STATEMENT
The authors confirm that the ethical policies of the journal, as noted on the journal's author guidelines page, have been adhered to, and this study was performed as stated by the Declaration of Helsinki principles. The Ethics Committee of the School of Veterinary Medicine, Shiraz University, certified all protocols (Register numbers: PHD891925). All volunteer human participants signed an informed consent.